Freeze-dried composition

ABSTRACT

The invention relates to the use of a polysaccharide having at least four saccharide units, such as stachyose, as a glass-forming agent for the freeze-drying of a reaction mixture comprising an enzyme. In particular, the enzyme is a polymerase useful in a nucleic acid amplification reaction such as a Polymerase Chain Reaction. Compositions comprising such polysaccharides as well as methods for preparing them, kits containing them and methods for using them form further aspects of the invention.

The present invention relates to freeze-dried compositions, inparticular those used to carry out chemical or biochemical reactions andcontaining a polymerase, as well as methods for preparing these andtheir use.

Lyophilization or freeze-drying is a well-known technique for preservinga wide range of moieties including chemicals, biochemicals andbiological samples. It is well known that these moieties need to bemixed with reagents such as stabilisers. A variety of stabilisers areknown and these are required to ensure that during the freeze dryingprocess, the moieties retain their structure and function.

In a particular instance, some or all of the reagents necessary forcarrying out specific chemical or biochemical reactions are combined andthe mixture frozen or freeze-dried. The resulting material can then bestored or transported to the laboratory or environment in which thereaction is later carried out, for example by addition of a sample whichmay for instance, be a clinical, environmental or other biologicalsample that requires testing. Freeze-dried material provides a moreconvenient form for storage and transportation as there is norequirement for refrigeration.

A particular reaction in which this procedure has been used is nucleicacid amplification methods such as the Polymerase Chain Reaction (PCR).In this reaction, specific nucleic acid sequences are multiplied oramplified by subjecting a sample containing that nucleic acid to aseries of iterative steps. Primers that are specifically designed tohybridise to the target nucleic acid sequence are included in thereaction and are extended by polymerase enzymes and in particularthermally stable polymerases present in the reaction mixture to producecopies of the target in an exponential manner. The progress of thereaction may be monitored in real time, using labelled probes or otherfluorescent moieties that are arranged to produce a variety of labellingsystems.

The target nucleic acid sequences amplified may be DNA or RNA. Where thetarget nucleic acid is DNA, the polymerase used will be a DNA dependentDNA polymerase. In the case of RNA being the target, the amplificationproduct is either the complementary cDNA or RNA and it is produced in aparticular variant of PCR known as reverse-transcriptase PCR or RT-PCR.This reaction uses either an RNA dependant DNA polymerase or an RNA/DNAdependant DNA polymerase as a reverse-transcriptase enzyme to producethe cDNA (covert RNA to RNA). As used herein, references to thepolymerase chain reaction or PCR include RT-PCR as well as conventionalPCR and the term ‘polymerase’ includes both DNA polymerase andreverse-transcriptase.

Whilst the primers and probes will be specific to particular reactions,there are other reagents, in particular polymerase enzymes, nucleotides,buffers and salts such as magnesium salts that are common to all suchreactions. These form elements of so-call ‘core master-mixes’ and theseare available ready mixed from commercial sources. These may be frozenor freeze-dried as discussed above, and so called ‘PCR-ready beads’ areavailable. Where specific PCRs are conducted on a regular basis, forinstance, for a particular diagnostic purpose, the primers and probes orother label reagents required may be incorporated into the freeze-driedreagent mixture so that they are ready for use directly on addition of asuitable sample.

Freeze-drying of such complex mixtures requires a careful considerationof the stabilising reagents that may be included. These are required toprotect the reagents and in particular the biologically active reagentssuch as the enzymes during the freeze-drying process, but they must notthen inhibit or affect the subsequent reaction that is carried out usingthe freeze-dried mixture.

Various stabilising agents have been suggested for use in this case. Forinstance US Patent No. 2002/0173016 suggests that a combination of a lowmolecular weight sugar (which may itself be a combination of di- andtrisaccharides) and a starch provides a suitable stabilizing compositionfor lyophilisation of polymerase containing reaction mixtures.WO2010/001162 teaches that the trisaccharide raffinose and in particularraffinose pentahydrate is a suitable glass-forming agent for use in suchreaction mixtures in particular where fluorescent reagents are alsopresent.

Although higher molecular weight sugars such as stachyose have been usedpreviously as a stabiliser in freeze-drying of some pharmaceuticalproteins (Prestrelski et al, Pharmaceutical Research, (1995) 12, 9 p1250-1259), these are generally thought to be less effective than lowermolecular weight sugars in relation to the freeze-drying of the enzymeβ-galactosidase (Yoshioka et al. Pharmaceutical Research (2007) 24, 9,1660-1667).

The applicants have found that higher molecular weight saccharides areparticularly beneficial glass-forming agents for use in freeze-drying,in particular of chemical and biochemical reaction mixtures comprising arange of enzymes, in particular those that are useful in themanipulation of nucleic acids such as polymerases.

According to the present invention there is provided the use of apolysaccharide having at least four saccharide units as a glass-formingagent for the freeze-drying of a reaction mixture comprising an enzymeother than β-galactosidase.

In particular, the enzyme is not an enzyme that breaks down sugars.

In a particular embodiment, the enzyme may be an enzyme that has aneffect on nucleic acid, protein or pyrophosphate. Such enzymes may beselected from the group consisting of polymerase, DNAse, RNAseexonucleases, nuclease inhibitors, restriction enzymes, uracil DNAglycosylase, protease or pyrophosphatase.

In a particular embodiment, the enzyme is a polymerase.

The polysaccharide suitably comprises from 4 to 8 and preferably from 4to 6 saccharide units. In particular, the saccharide is selected fromstachyose, verbascose and lycopose. A particularly preferred saccharideis stachyose.

Whilst pure saccharide may be used, a good source of saccharide materialare natural product extracts that may contain a variety of saccharidesincluding some which have less than 4 saccharide units. However,provided a substantial portion of the saccharides present, in particularat least 50%, such as at least 60%, 70%, 80%, 90% or 95% have at least 4saccharide units, a beneficial effect can be found.

In particular, by using the higher molecular weight sugars such asstachyose as the major stabilizing element of the mixture, theapplicants have found that freeze-dried compositions obtained haveimproved resistance to hydration on exposure to moisture in for examplethe air. It would appear that the sugar is acting as a more effectivedessicant, and this increases the longevity of the composition.

However, in addition, compositions containing these sugars can bedesigned to have better dissolution properties than formulationscontaining lower molecular weight sugars, as the amount of other highmolecular weight excipients that tend to reduce solubility, can bereduced. In addition, the sugars solutions have a longer shelf life thansimilar solutions of lower molecular weight sugars making them easier tohandle and store during the manufacturing process. Furthermore, sugarssuch as stachyose gets into solution better than raffinose, whichrequires gentle warming to get it into solution. Furthermore, raffinosehas a tendency to precipitate out when stored in solution at lowtemperatures as are found in a refrigerator, whereas stachyose and someother high molecular weight sugars do not. This means that solutionsderived from compositions containing stachyose may be easily stored in afridge and show reduced spoilage as a result of bacterial contamination.

In particular, according to a further aspect of the present inventionthere is provided a composition comprising:

(i) an enzyme other than β-galactosidase;

(ii) a polysaccharide comprising at least 4 saccharide units.

The composition is suitably in a freeze-dried form. Alternatively, it isin the form of a solution ready for freeze-drying or as a solutionreconstituted from said freeze-dried form.

The enzyme may be any of the enzymes listed above, but is suitably anenzyme that impacts on nucleic acid such as a polymerase. A variety ofpolymerases are known and these selected depending upon their intendedpurpose in a reaction. For instance, there are a range of isothermalamplification reactions such as the transcription mediatedamplification, nucleic acid sequence-based amplification, signalmediated amplification of RNA technology, strand displacementamplification, rolling circle amplification, loop-mediated isothermalamplification of DNA (LAMP), isothermal multiple displacementamplification, helicase-dependent amplification, single primerisothermal amplification, and circular helicase-dependent amplificationas well as whole genome amplification, where the polymerase used needonly be active at the temperature at which the reaction is carried out.

In a particular embodiment, the polymerase is a thermostable polymerasesuch a Taq polymerase or Pfu or a reverse-transcriptase enzyme such asMMuLV, AMV or Tth that is used in reactions such as the polymerase chainreaction which involve thermal cycling, including stages carried out athigh temperatures. The polymerases may be recombinant to form enhancedthermostability or processivity, or other property, for example they mayinclude fusion proteins combining DNA binding domains with a polymerase.The thermostable polymerase maybe chemically modified or otherwiseblocked so that it is inactivated until subject to an initial heatactivation step, as used in a conventional ‘HotStart’ PCR. Such enzymesare available from a variety of commercial sources and are described forexample in U.S. Pat. No. 5,677,152. The polymerase may also form anantigen to antibody conjugate (Taq and anti-Taq antibody) as tofacilitate a hot start as described for example in U.S. Pat. No.5,338,671 or U.S. Pat. No. 5,586,287). Other modifications orco-reactant species used to facilitate a hotstart such as aptamers,peptides or other species.

The polymerase enzyme is suitably present at a concentration that willallow it to carry out a reaction such as a PCR when the composition ofthe invention in freeze-dried form is reconstituted. This will dependupon factors such as the volume of the freeze-dried composition used andthe volume of the reconstituted reaction mixture. However, in generalpolymerase is present in a reaction such as a PCR in concentrations offrom 0.01 units/uL (10000 UNITS/L) to 0.2 units/uL (200000 UNITS/L) andthis may typically be achieved by ensuring that the freeze-driedcomposition contains from 10000-200000 units/L in particular from 40000units/L where a ‘unit’ is defined as the amount of enzyme that willincorporate 15 nmol of dNTP into acid-insoluble material in 30 minutesat 75° C.

As set out above, the saccharide suitably has from 4 to 8 and preferablyfrom 4 to 6 saccharide units. In particular, the saccharide is selectedfrom stachyose, verbascose and lycopose. A particularly preferredsaccharide is stachyose. The polysaccharide may comprise a mixture ofsaccharides such as found in an extract from a natural product. There isno need however for the addition of starch or other components.

The polysaccharide must be present in a glass-forming amount in thecomposition. This will vary depending upon the other components presentin the mixture. Where the salt content of a reaction mixture is high,the levels of polysaccharide may need to be increased to achieve thedesired protective effect. However, the polysaccharide should not bepresent in such a significant amount that upon reconstitution of thecomposition by addition of water to produce a “final composition” foruse in a chemical or biochemical reaction, it is present in such anamount that it inhibits or otherwise restricts the reaction. The amountin a final composition is typically no more that 10% m/v for examplefrom 0.5 to 10% m/v and suitably from 1-20% m/V for the solution used toform the lyophilised glass substrate. The relative volume of thecomposition before and after freeze-drying will vary depending upon theamount of water required to solubilise the components to form theadmixed composition. In general however, the volume of the compositionpre-lyophilisation will be from 0.1 to 0.5 times the volume of thecomposition after lyophilisation and dissolution/reconstitution, andsuitably about half the volume. This means that typically, the amount ofsugar present in the freeze-dried composition or ‘cake’ is from 0.5 to5% m/v for instance about 2.5% m/v.

The composition may further comprise additional components required inorder to carry out a chemical or biochemical reaction such as thepolymerase chain reaction. In particular, the reaction mixtures mayfurther comprise a buffer used in the PCR reaction, in particular abuffer having a pH of from 7-9 for instance from 8 to 9. Suitablebuffers include Tris or Trizma buffers as well as HEPES, tricine andbicine buffers in some cases. Where the buffer is not included in thecomposition, it would be necessary to ensure that a suitable rehydrationbuffer is used to reconstitute the dried composition ready for use. Theamount of buffer present in the composition will be such as to ensurethat when the freeze-dried composition is reconstituted, it will producea concentration of buffer in the final reaction mixture in accordancewith that which is conventional in the art, depending upon theparticular buffer employed. For instance, Tris buffer concentrations inthe range of from 1-50 mMol/l may be used, such as from 5-35 mMol/L andin particular from 10-20 mMol/L are generally required in finalamplification reaction mixtures and so appropriate concentrations willbe added to the composition before lyophilisation.

Similarly the composition may further comprise salts required for use inthe reaction. In the case of PCR, such salts will be for examplemagnesium, sodium, lithium, potassium, ammonium or manganese salts, suchas halide salts for example chlorides or sulphates. A particular saltthat is used in this way is magnesium chloride (MgCl₂). Salts will bepresent in an amount that is necessary for carrying out the reaction.Thus in the case of polymerase chain reaction, salts and in particularmagnesium salts may be present in the freeze-dried composition in anamount of from 1-10 mM and suitably from 1.5-6 mM such as from 3-6 mM toproduce suitable salt concentrations on reconstitution. However, it hasbeen reported that the presence of salts can lead to instability in thecomposition and so again the necessary salts may be omitted from thecomposition and added subsequently with the rehydration buffer ifrequired. In this embodiment, the salts may be included in a kitcomprising the composition and the necessary salt supplements.

Alternatively, the salts such as magnesium may be present but inconcentrations which are lower than are required for use in thereaction, for example at concentrations of less than 500 μM. Asdescribed in WO2006/003439, it has been found that such small amounts ofmagnesium salts may in fact be beneficial to the stability of thecomposition. In this case, supplementary salts will be included in therehydration buffer.

Generally, compositions used for reactions such as the polymerase chainreaction will include the nucleotides that form the building blocks ofthe amplification product. These may include deoxy or deaza nucleotidescontaining GATC or U nucleosides or their derivatives. In some cases,the nucleotides themselves will be fluorescently labelled. These areavailable from a variety of commercial sources. They will be present inthe freeze-dried composition in a concentration that will provide asuitable concentration of nucleotides in the reaction mixturereconstituted from it. This will depend upon the volume of thefreeze-dried composition and the final volume of the reaction mixturereconstituted from it. Typically, a PCR requires a concentration of eachnucleotide of from 50 μM to 8000 μM and this may generally be achievedby including nucleotides in the composition of the invention infreeze-dried form of from 0.1 to 1 mM, for example from 0.2 to 0.4 mM.

In a particular embodiment, a blocking compound, as is conventional inPCR reaction mixtures, may be included in the composition. The blockingcompound is believed to function by preventing inhibition of the PCR byinteraction with the vessel walls, for example by preventing leaching ofmetals or sequestering any metals which may leach from the walls in thecourse of the reaction. It may also reduce abstraction of enzyme andnucleotides to the reaction vessel wall. The nature of the blockingcompound will depend upon the nature of the vessel into which it isintended that the reaction should be conducted.

Particular examples of blocking compounds are glass coating or glassblocking compounds such as bovine serum albumin (BSA) either alone or incombination with other blocking materials such as gelatine. Gelatine maybe obtained from a variety of sources including bovine, pig, seaweed(carrageenan) or fish gelatine as described in WO2008155524.

Blocking agents are suitably included in effective amounts which willdepend upon the particular compound selected. However, for BSA forinstance, the amount is suitably sufficient to provide from 0.1 to 1mg/ml and preferably about 0.25 mg/ml in the final reaction composition(i.e. the composition made up for carrying out the chemical orbiochemical reaction). Gelatines will suitably be present in an amountin the range of from about 0.0025%-0.01% m/v or about 0.0025%-0.01% w/w.Care should be taken that the amount of blocking agent is notsufficiently high so as to significantly inhibit the final reaction.

The compositions suitably further comprise an anti-oxidant and/oranti-maillard reagent. The applicants have found that threonine works asa particularly effective anti-oxidant and/or anti-maillard agent, andenhances the stability of the freeze-dried composition. Either L- orD-threonine is used. The threonine appears to react with any oxygenproduced, or as a result of ingress into the resulting product andtherefore assists in the stabilisation of the mixture.

Furthermore, it has been found that the presence of threonine maystabilise the signalling achievable from fluorescent labels included inthe composition, in particular when stored at elevated temperatures.

The amount of threonine in the composition will vary depending upon theprecise nature of the composition. It is suitably selected so that itdoes not affect the pH of the composition, which may be important insome chemical or biochemical reactions. Typically however, it may bepresent in the final reaction composition in an amount of from 1-20mM/L, for example at about 5 mM/L. As discussed above, this means thatthe threonine will be present at a higher concentration, for example, atapproximately twice these concentrations in the freeze-driedcompositions.

When a composition is freeze dried in the presence of a glass-formingreagent, it generally forms a “cake” type 3-dimensional structure. Thisstructure is optionally supported by the inclusion of a suitablestabiliser for the cake structure, and so this is a further component ofthe mixture.

Examples of suitable stabilisers that may be included in the compositioninclude polymeric compounds such as polyethylene glycol (PEG),polyvinylpyrrolidine (PVP) and or polysaccharides such as Ficoll orDextran. In a particular embodiment, however, the stabiliser is omittedor used at reduced concentrations within the composition as it has beenfound that compounds such as PEG may contribute to the inhibition offluorescent signals. It may also reduce solubility of the overallcompositions described above. In the context of the compositions of thepresent invention, the use of high molecular weight sugars as theglass-forming agent means that the amount of stabiliser used in thefinal composition may be less than 4% M/v, for instance less than 1% M/vfor example less than 0.8% for 0.1-0.5% M/v such as about 0.25 M/v %. Inthe freeze dried compositions and cake therefore, there will typicallybe less than 2% M/v, for example from 0.2 M/v to 1% M/v for instanceonly about 0.5% M/v stabliser present.

As mentioned above, the precise enzymes and other reagents present willbe selected depending upon the particular nature of the chemical orbiochemical reaction being effected. They may include reactions carriedout on multiple or repeated occasions such as diagnostic tests,screening tests, nucleic acid amplification reactions, nucleic acidsequencing reactions etc.

In addition, where the polymerase enzyme is not chemically or otherwise‘blocked’ as discussed above, alternative ‘Hot Start’ technologies maybe incorporated into the composition by inclusion of suitable additioncomponents. For instance, it is possible to add an anti-Taq DNApolymerase antibody such as those available from Clontech, Sigma orInvitrogen, as a further component of the composition. Such antibodiesbind to the active site of the polymerase and inactivate it at ambienttemperature to form an antigen to antibody conjugate of the typediscussed above. However, the antibody denatures and dissociates fromthe enzyme at elevated temperatures used during the amplification cyclesand so the enzyme becomes active. The relative amount of any anti-Taqantibody included in the composition is suitably sufficient to ensurethat it is able to fulfill the function of inhibiting the Taq enzymeuntil it is required. Generally therefore an excess of anti-Taq antibodyas compared to Taq enzyme will be used. Thus for example for every unitof Taq enzyme in the composition, at least 1.5 and preferably at least 2units of anti-Taq antibody will be included. Taq antibody is usuallysold by the μg and the concentration is very dependent upon the sourceand quality of the antibody as well as the nature of the assay. Too muchantibody may be detrimental and can actually cause more primer dimer insome assays. However, the precise amount of Taq antibody will bedetermined in accordance with usual practice and will typically be inthe range of 0.001 to 0.004 μg/l final reaction mixture.

Yet another Hot-Start methodology involving the use of a combination ofan inhibitory amount of a pyrophosphate salt to prevent primer extensiontaking place, and a pyrophosphatase enzyme which digests thispyrophosphate at elevated temperatures, to allow the PCR to progress isdescribed in WO 02/088387.

In this case, the pyrophosphate salt and the pyrophosphatase enzyme maybe included as further components of the composition of the invention.

If required, the composition may comprise further elements that may beuseful in the reaction, such as the PCR. A particular example of such anelement may comprise a nucleic acid that is able to act as an internalcontrol for the amplification reaction. This nucleic acid may be a‘homologous’ nucleic acid which is recognized by the same primers as thetarget nucleic acid in the sample, but which produces different anddistinguishable product, for example of a different size. Alternatively,it may be heterologous nucleic acid which is amplified by a differentset of primers, which are, in that case also included in thecomposition, so as to yield a different product which is clearlydistinguishable from the amplification target. In either case, thepresence of the product of amplification of the nucleic acid acting asinternal control will provide confirmation that the amplificationconditions have been effective and that therefore amplification oftarget, if present, would be expected.

The enzyme such as the polymerase used in the composition is, asdiscussed above, obtainable from a variety of sources. Generallyhowever, as supplied, the enzyme is in the form of a wet reagent whichincludes a range of excipients including detergents, anti-oxidants,anti-reducing agents and significant quantities of solvent which isgenerally glycerol but in some cases, oligosaccharide solutions areused. For example, some polymerase enzymes are supplied as a 50%glycerol solution, the latter being present to assist withfreeze-thawing carried out during the enzyme production and storageprocess. Removal of the glycerol is necessary to effect an efficientfreeze-drying process however. The applicants have found thatsubstantially complete removal of glycerol is beneficial to thefreeze-drying process so that the composition is substantially free ofglycerol. In particular the composition should contain less than 1% v/vglycerol, for example less than 0.5%, 0.22%, 0.11% or 0.01% vv glycerol.Suitably, the composition is free of glycerol.

Removal of glycerol from commercial polymerase preparations can beeffected using a variety of conventional techniques including forinstance separation based upon molecular weight, such as dialysis,microfiltration using a membrane or exclusion chromatography forinstance on a Sepharose™ column, or separation based upon affinitytechniques such as ligand binding, histadine tagging, or by usingspecific binding partners such as antibodies or aptamers.

In a particular embodiment, one or more washing steps using a buffersuch as a Tris buffer are carried out during or subsequent to theremoval procedure to ensure substantially complete removal of glycerol.

At this stage, it may be necessary to re-introduce the other agents suchas detergent, anti-oxidant and anti-reducing agent that facilitate theactivity of the enzyme during the reaction since these components may belost during such a thorough removal procedure. Generally, the specificreagents which tailor a PCR to the particular target, such as theprimers and any probes required for example for use in connection with areal-time PCR, are specific to a particular area of application such asresearch and so added when the freeze-dried composition is reconstitutedinto a reaction mixture for use. However, in many cases, in particularin the diagnostics field, the targets are the same in many cases, andtherefore the inclusion of oligonucleotides that act as probes andprimers into the freeze-dried composition is convenient, so that thecomposition becomes assay specific is desirable for ease of use.

Alternatively, one or more separate compositions comprisingoligonucleotides that act as probes and/or primers as well as salts orbuffer compositions required to carry out the reaction, may be preparedseparately and conveniently these may also be dried. They may befreeze-dried but in some cases, they may be dried using other methodssuch as air drying, or dried by direct application of heat to causeevaporation of the solvent (e.g. water, ethanol), rather thansublimation as occurs in freeze drying.

For example, using this approach, buffers or salts including potentialenzyme cofactors, that may be required for the final reaction, but whichcould be inhibitory, for example, to the stability of the polymerasesduring and post drying to be stored separately from the enzymes beforeuse. Examples of such buffers or salts may include ammonium sulphate butthere are others as would be appreciated by the skilled person. Anexample of an enzyme cofactor which may be dried and stored separatelyis magnesium or manganese ions in the form of salts such as magnesiumchloride. Such enzyme co-factors could be dried separately to form anadditional or alternative separate compositions, or conveniently, thesemay be included in the dried buffer composition as a single secondcomposition. Keeping the buffer components and/or co-factors separate inthis way suppressed the polymerase activity of the enzyme until it isre-suspended using the reconstitution buffer, thus further improving thestability of the composition.

Such additional compositions may be re-hydrated by the end-user beforeuse. Depending upon the content of the second composition, they may bereconstituted using a solution of any residual components required inthe reaction, for example using a cocktail of primers and probes insolution that are required to complete their specific amplificationreaction, or a reconstitution buffer solution if the buffer componentsare not already included in the composition.

However, in a particular embodiment, it may be convenient to provideprobe and/or primer compositions in dried form and separately, inparticular in kits where a single mastermix may be required to be usedin a variety of different amplification reactions.

In such cases, where the second composition is freeze-dried, it may beconvenient, although not essential, to utilise similar glass-formingagents as those described above. The applicants have found thatpolysaccharides such as stachyose provide effective glass-forming agentsfor such compositions including oligonucleotide compositions asexemplified below. Thus, in a further aspect the invention provides akit comprising the composition of the invention and a second compositionin dried form, for example in freeze-dried form, said second compositioncomprising a reagent, such as an oligonucleotide useful as a probe orprimer in a nucleic acid amplification reaction and/or buffer reagentssuch as salts, for use in a reaction with said enzyme. The secondcomposition may further comprise stachyose as described above.

Novel compositions such as dried buffer compositions that may beincluded in said kits as said second compositions form a further aspectof the invention. Thus, in a further aspect, the invention provides adried composition comprising one or more of the following:

(i) a salt required to carry out a nucleic acid amplification reaction;

(ii) components of a reconstitution buffer useful in an amplificationreaction;

(iii) an oligonucleotide capable of acting as a primer in a nucleic acidamplification reaction: and

(iv) an oligonucleotide capable of acting as a probe in a nucleic acidamplification reaction.

Any oligonucleotides used in the compositions or second compositions asdescribed above may be labelled as is well known, using for instance afluorescent label. The composition may further comprise a labelledoligonucleotide, such as a fluorescently labelled oligonucleotide(s)useful in monitoring the progress of a polymerase chain reaction in realtime. Other fluorescent agents useful in detection of reactions such asPCR in ‘real-time’ such as intercalating fluorescent dyes may also beincluded in the composition. In other assays such as the Plexor® assay,fluorescent mononucleotides are used in the monitoring process and ifthe composition is intended for use in such an assay, the labellednucleotides may be included in the composition of the invention.

The amounts of the various components included in the composition willvary depending upon factors such as the precise nature of the particularcomponent; the nature of the PCR which it is intended should beconducted etc. However, this will be determinable in each case usingestablished protocols and procedures as would be understood in the art.

Compositions of the invention are suitably freeze-dried to form a driedcake that may be distributed in the form of pellets and/or beads. In aparticular embodiment, sufficient composition to carry out multiplereactions, for example up to 96 or 48 reactions are freeze-dried to forma single large cake in a container. The container should be ofsufficient size to allow sufficient water or solvent to be added toreconstitute sufficient solution to effect multiple reactions.Individual reaction mixture volumes may then be dispensed from thecontainer for example using a pipette, to the reaction vessels in whichthe reaction is to be carried out. Such reaction vessels may include thewells of microtitre plates.

In a particular embodiment however, the compositions of the inventionare formed in solution form by mixing the reagents in water, and thenfreeze-dried in situ to form a cake structure.

In another embodiment the cake structure may be formed from two or morelayers of different reagent formulations. The purpose of this may be toenhance either the resulting cake stability and/or the performance ofthe final reaction upon dissolution. The active formulation of manyreactions may compromise the optimum stability of one or more specificcomponents. One or more of these components may be included in aseparate reagent layer.

For example, certain fluorophores have a greater stability at particularpH's. Fluorescein derivatives prefer basic environments, whereas,cyanine derivatives prefer neutral pH's which are not consistent withthe final amplification optimum range described above. Thereforestandard cakes prepared at pH 8-9 would compromise the stability ofcyanine fluorophores.

Likewise, polymerases may be delineated into their own layer such thatthey are not available to interact with specific reaction componentssuch as primers or probes. This would provide a pseudo ‘hotstart’ duringthe lyophilisation process, the resulting reaction only becoming activeafter dissolution of both layers.

Each layer may contain one or more of the reagents from the finalamplification formulation together a glass-forming sugar as describedabove, and any other excipients such as the stabilizer. The layers areformed by dispensing reagents individually and freezing each one beforedispensing the next reagent thereover. The resulting structure of frozenlayers can then be freeze dried. The precise formulations of each layerwould be formulated as to provide a suitable final composition uponco-dissolution, whilst drying to form a single cake structure.

In summary then, layered compositions of this type may be prepared by aprocess comprising (i) freezing a solution of a polysaccharide asdefined above and either one or more of said further components or saidenzyme to form a first frozen layer, (ii) adding a second solution of apolysaccharide as defined in claim 1 and a different one of said furthercomponents or said enzyme and freezing the second solution into adistinct layer, (iii) optionally repeating step (ii) until allcomponents of the composition are present in one of the frozen layers,and (v) freeze-drying the resultant structure. The cake obtained in thisway will also have a layered structure, allowing components of themixture which are incompatible in some way to be held separately in thecake so that they do not interact significantly before the cake isdissolved for use.

In a particular embodiment, the compositions of the invention arefreeze-dried in a reaction vessel suitable for carrying out the ultimatereaction such as the polymerase chain reaction. This means that thecompositions may be reconstituted and reacted in the same vessel,avoiding the need to transfer reagents between vessels and so avoidingsome contamination risks.

A particularly suitable vessel for holding compositions as describedabove is as described in co-pending British Patent Application No.1208808.4. It comprises thermally conducting plastics that allow rapidtransfer of thermal energy from the contents during freeze-drying andalso rapid transfer of thermal energy during the heating and coolingcycles required for carrying out a PCR. Such vessels may be in the formof single tubes, or multi-well plates such as microtitre plates thatconventionally contain 96 individual wells.

Thus such vessels, preloaded with the compositions of the invention infreeze-dried form can be stored, transported and sold ready for use.

In use, the freeze-dried composition is reconstituted by addition of asample in liquid form together with any components that are required forthe reaction but are not present in the composition as discussed above.The thus formed solution is then subjected to appropriate reactionconditions, which, in the case of PCR, is a thermal cycling procedure.

Methods for preparing the composition of the invention and the usethereof form further aspects of the invention.

Thus, in a further aspect, the invention provides a method for preparinga composition as described above in freeze-dried form, which methodcomprises combining together a polymerase enzyme, a polysaccharide asdefined above and optionally one or more of the further components asdescribed above in solution form, and freeze-drying said mixture.Suitably, in a preliminary step, glycerol is substantially completelyremoved from the polymerase enzyme used as described above.

In a particular embodiment, the liquid composition is divided amongstmultiple reaction vessels, which suitably comprise a thermallyconducting plastic as described above, so that each vessel containssufficient reagents to carry out a single reaction using the polymeraseenzyme, and freeze-dried within said vessels.

In a further aspect, the invention provides a method for carrying out achemical or biochemical reaction using a polymerase enzyme, said methodcomprising forming a reaction mixture by addition of a liquid and anyaddition components necessary for carrying out said reaction, to acomposition as described above in freeze-dried form, and subjecting thethus formed solution to suitable reaction conditions. In particular, thereaction will be a polymerase chain reaction. The use of thepolysaccharide as described above provides for excellent storage,stability and dissolution properties of the freeze-dried composition.

The invention will now be particularly described by way of example withreference to the accompanying drawings, in which:

FIG. 1 show the results of a stability trial carried out on a variety offreeze-dried sugar solutions;

FIG. 2 shows the results of PCR reactions carried out using reactionmixtures in accordance with the invention and fresh undried reactionmixture; and

FIGS. 3-5 shows the results of amplification reactions carried out usingvarious PCR enzymes which had been incorporated into compositions inaccordance with the invention.

EXAMPLE 1 Preparation of Compositions of the Invention

Freeze dried compositions of the invention are prepared by mixingtogether the components required for the reaction including enzymes andoptionally other components discussed above, with a sugar as definedabove such as stachyose in a glass-forming amount and a small quantity(less than 1% M/v and preferably less than 0.5% M/v) of a structuralstabiliser such as PEG, also as described above. Any enzyme used shouldbe substantially free of glycerol. At this stage, the mixture willcontain a solvent and in particular water. The amount of water presentis selected to give the desired cake volume after freeze drying.Specifically, during a freeze drying process, the mixture is firstfrozen, which effectively determines the volume of the cake. Duringsubsequent sublimation of the solvent, the frozen material may undergosome shrinkage, but will generally retain a substantial proportion ofthe volume of the frozen material. This is useful in that it allows acake of a suitable volume to be produced. Thus the amount of solventsuch as water present in the mixture at this time will vary dependingupon factors such as the amount required for dissolution of thecomponents and the desired cake volume. Typically, any solution suitablycontains less than 50% water as solvent compared to the finalreconstituted reaction composition.

The solution is then freeze-dried to remove substantially all of thewater in a freeze dryer used in accordance with the manufacturer'sinstructions. The freeze-dried composition forms a cohesive andmalleable cake.

In particular, the or each container holding the freeze-driedcomposition is sealed under an inert atmosphere such as a nitrogenatmosphere.

Just prior to use, the freeze-dried compositions are reconstituted byaddition of a suitable volume of water which may include a sample undertest. The reaction mixture is then processed in the usual way.

It has been found that the freeze-dried compositions of the inventionare more stable for prolonged periods, for example in high humidityprior to use, when compared to published methods. They also reconstitutemore easily, after exposure to form active reaction mixtures which areextremely reliable in use.

EXAMPLE 2 Comparative Environmental Exposure Properties of Freeze-DriedCakes of Various Sugars

The following solutions were prepared:

A) 2.5% (m/v) Raffinose, 0.5% (m/v) PEG, 500 uL cakeB) 2.5% (m/v) Stachyose, 0.5% (m/v) PEG, 500 uL cakeC) 2.5% (m/v) Trehalose, 0.5% (m/v) PEG, 500 uL cakeThese were each freeze dried to form a similar 5004 cake. The cakes werestored in a desiccator at about 75% humidity in the presence of asaturated solution of sodium chloride at ambient temperature. Thestability of the cakes over time was monitored and the results areshowed that the stachyose composition (B) retained stability better thanboth compositions (A) and (C).

The experiment was repeated using slightly larger (5254) cakes. Thematerials were placed in glass lyovials, and then dried using a VirtisAdvantage+ freeze drier, operating a lyophilisation cycle comprising athermal treatment step, a primary drying step and a Post hold step assummarised in the following tables:

Thermal Treatment Step No Temp° C. Time (minutes) Ramp/Hold 1 10 15 Hold2 −45 110 Ramp 3 −45 120 Hold

Primary Drying Step No Temp° C. Time (minutes) Vacuum Ramp/Hold 1 −45900 100 Hold 2 0 120 100 Ramp 3 0 60 100 Hold 4 20 50 100 Ramp 5 20 120100 Hold

Post hold Temp° C. Time (minutes) Vacuum 20 1000 100Condenser temperature: −55° C.

Secondary Set Point: 45° C.

A bell jar was filled with a saturated sodium chloride solution atambient temperature to produce an approximate relative humidityenvironment of ^(˜)75%. The caps from the three lyovials were removedimmediately before placement within, and closure of, the bell jar. Thehydration of the cakes was filmed using time lapse photography.

The results are shown in FIG. 1. These show that the stachyose cake (B)was resistant to collapse showing marginal alteration in cake structurewithin the period of the experiment (^(˜)80 time minutes). The raffinosecake (A) showed almost complete collapse after ^(˜)65 minutes. Thetrehalose cake (C) showed approximately 50% collapse at the completionpoint of the experiment^(˜) (^(˜)80 time minutes) minutes.

As a result, stachyose appears to provide a more stable freeze driedcomposition than either raffinose or trehalose.

EXAMPLE 3 Generation and Testing of a Lyophilised PCR Mastermix

This example illustrates the generation of a lyophilised core master mixpreparation according to the invention. This includes the use of achemically modified hot start polymerase Taq DNA dependent polymerase ina suitable buffer composition dried in a glass lyovial.

Example of freeze dried master mix composition:

Reagent Cake Concentration (2x) Final Concentration (1x) Tris 20 mM 10mM MgCl2 6 mM 3 mM BSA 500 ng/μl 250 ng/μl dNTPs with dUTPS 0.4 mM 0.2mM Stachyose 2.5% 1.25% PEG 0.5% 0.25% TAQ polymerase 0.08 U/μl 0.04U/μl TAQ additive* ≡0.08 U/μl ≡0.04 U/μl *TAQ additive is a detergentmix obtainable from Fluorogenics Ltd.

A cake comprising the above components was prepared and was freeze driedin a Virtis Advantage+ freeze drier using the program described inExample 2 to create active lyophilised product having a finalconcentration as listed above.

The resulting freeze dried mixture was reconstituted by addition ofvarious concentrations of water as detailed below and used to amplify aDNA target (derived from bacteriophage λ) using custom primers incombination with SYBR® Green-1 DNA binding dye to a final concentrationof 1:10,000 of reference solution. The mixture was amplified on theGenie thermal cycler using:

PCR Parameters

Denature 95° C. 900 seconds Amplification 95° C. 20 seconds 55° C. 20seconds 74° C. 20 seconds Optical Read X5 45 Cycles Melt 50° C. 20seconds 95° C. 10 seconds Continuous read −1 Rate = 0.1° C./second

The resulting mixture was used to amplify a DNA target (derived frombacteriophage A) using custom primers in combination with SYBR® Green-1DNA binding dye to a final concentration of 1:10,000 of referencesolution. The data (FIG. 2) shows three 10-fold dilutions (labelled 10̂5,10̂4, & 10̂3) of template and a no template control in duplicateamplifications. The solid lines are reactions prepared using lyophilisedmaster mix. The dotted line shows the same formulation prepared as anon-lyophilised control.

The results are substantially equivalent showing that the stachyose hasprotected the activity of the active components through thefreeze-drying process.

EXAMPLE 4 Preparation and Use of a Two-Part Composition Kit

A lyophilised core PCR master mix was prepared according to theinvention, and a second composition comprising a lyophilised primer andprobe mixture was also prepared. The Taq enzyme used in this example wasan anti-Taq antibody mediated hot start Taq DNA dependent polymerase ina suitable buffer composition, available from Promega (USA).

Primer Probe Recipe (2× cake formulation), dried to 12.5 μL volumefinal.

REAGENT CAKE CONCENTRATION (2x) Units HSV 1 F 1 uM HSV 1 R 1 uM HSV 1 P0.4 uM HSV 2 F 1.8 uM HSV 2 R 1.8 uM HSV 2 P 0.8 uM Trizma 2 mMSTACHYOSE 2.5 % M/V PEG 0.5 % M/VMaster Mix Recipe (2.5× cake formulation) dried to 5 μL volume final(1×).

CAKE CONCENTRATION REAGENT (2.5 x) MgCl2 10 mMols/L BSA 625 mg/LdUTPS(dUTP/dNTP mixture) 0.5 mMols/L Taq 100000 UNITS/L Trizma 25mMols/L STACHYOSE 2.5 % (M/v) PEG 0.5 % (M/v)

Both compositions were freeze dried as described in Example 2, but inseparate micro-amp style vessels.

The resulting compositions were used to amplify a DNA target (derivedfrom HSV 1 and 2) using custom primers in combination with dual labelledfluorogenic probes for each target using the 5′ nuclease assay process.

The primer probe pots were resuspended in 12.5 μL of water to which 12.5μL of either template or water (for no amplification controls) was addedand mixed. 12.5 μL was transferred to the core master mix pot and mixedbefore transfer to an ECO plate. The mixture was amplified on theIllumina ECO thermal cycler using the following parameters:

PCR Parameters

Denature 98° C. 600 seconds Amplification 95° C. 5 seconds 62° C. 35seconds, Optical Read in FAM and HEX channels 45 CyclesThe results are shown in FIG. 3. They illustrate efficient amplificationfor both templates tested.

EXAMPLE 5 Generation and Testing of Other Lyophilised PCR ReactionMixtures

This example illustrates the generation and use of a lyophilised coremaster mix preparations according to the invention.

A first mastermix included as the enzyme a high fidelity recombinantpolymerase Pfu DNA dependent DNA polymerase in a suitable buffercomposition.

Reagent Cake Concentration (2X) Final Concentration Trizma 20 mM 10 mMBSA 500 ng/μl 250 ng/μl dUTP/dNTP 0.4 mM 0.2 mM Pfu enzyme 0.04 U/μl0.02 U/μl Stachyose 2.5% 1.25% PEG 0.5% 0.25%

The composition was freeze dried as described in Example 2 in a glasslyovial. The resulting mixture was resuspended in resuspension bufferthat was able to provide dissolution and an active complete PCR reactionmixture. This mixture was then used to amplify a DNA target (derivedfrom bacteriophage A) using custom primers in combination with SYBR®Green-1 DNA binding dye to a final concentration of 1:10,000 ofreference solution. The mixture was amplified on the Genie thermalcycler using:

PCR Parameters

Denature 98° C. 30 seconds Amplification 98° C. 10 seconds 55° C. 30seconds 72° C. 30 seconds Optical Read X5 45 Cycles Melt 50° C. 20seconds 95° C. 10 seconds Continuous read −1 Rate = 0.1° C./second

The data shows three 10-fold dilutions (labelled 10̂5, 10̂4, & 10̂3) oftemplate and a no-template control in duplicate amplifications. Theresults illustrated in FIG. 4 show efficient amplification across thetested target template concentrations.

In a second experiment, a chemically modified hot start polymerase TaqDNA dependent polymerase in combination with a thermo-stable MMuLV RNAdependant DNA

RNase H-polymerase was used as the enzyme. This was combined with otherreagents including salts and appropriate concentrations of stachyose toform a PCR mastermix. This was dried in a glass lyovial using a methodas described in Example 2.

The resulting mixture was used to amplify a RNA target (derived from rat18s RNA) using custom primers in combination with dual labelledfluorogenic probes for each target using the 5′ nuclease assay process.The mixture was amplified on the Genie thermal cycler using:

PCR Parameters

RT-Step 48° C. 600 seconds Denature 95° C. 900 seconds Amplification 95°C. 20 seconds 60° C. 60 seconds Optical Read X10 45 Cycles

The amplifications were carried out at three 10-fold dilutions (labelled4.5 ng (per reaction), 0.45 ng (per reaction), 0.045 ng (per reaction)of template and a no-template control in duplicate amplifications. Theresults are shown in FIG. 5 solid lines are reactions prepared usinglyophilised master mix. The dotted line shows a commercial 2× master mix(non-lyophilised) control. The results are substantially equivalent.

EXAMPLE 6 Alternative Separate Compositions

As described above, one embodiment of the invention includes the use ofa core lyophilised PCR master with a lyophilised resuspension buffer,which forms a second composition. This resuspension buffer may also belyophilised using the same freeze drying procedure as the PCR master,but in a separate vial (although alternative drying techniques may beused as described above). This means that salts required for the buffercomposition in the final reaction, but which could be inhibitory, forexample, to the stability of the polymerases during and post drying tobe kept away from the enzymes until required. Likewise some of theenzyme cofactor, which in this case is magnesium ions in the form ofmagnesium chloride may be dried in this buffer.

This is illustrated here by an example of the use of an anti-Taqantibody mediated hot start Taq DNA dependent polymerase lyophilisedmaster mix for use in combination with the reconstitution buffer. Thereconstitution buffer mixture and lyophilised PCR master is dried usingthe same process but in separate vessels.

Reconstitution buffer cake recipe Recipe (2× cake formulation), dried to12.5 μL volume final.

REAGENT CAKE CONCENTRATION (2x) Magnesium Chloride 4 mMols/L AmmoniumSulphate 20 mMols/L Potassium Chloride 20 mMols/L STACHYOSE 2.5 % M/VPEG 0.5 % M/VPCR Master Mix Recipe (2× cake formulation) dried to 12.5 μL volumefinal (2×). The final PCR reaction would be 25 μL.

REAGENT CAKE CONCENTRATION (2 x) MgCl2 6 mMols/L BSA 500 mg/L dUTPS(dUTP/dNTP 0.4 mMols/L mixture) Taq 80000 UNITS/L Trizma 20 mMols/LSTACHYOSE 2.5 % (M/v) PEG 0.5 % (M/v)

The two separate dried compositions as set out above, may be collectedtogether in a kit.

A number of reconstitution regimes and solute permutations are possible.For example, in a first embodiment, the reconstitution buffer cake isre-suspended using only water to a 2× composition. The resulting mixtureis used to effect dissolution of the lyophilised master mix to a 2×solution. The complete solution will form a 2× mixture that may befurther diluted with primers, probes and template to carry out a PCR.

Alternatively, the reconstitution buffer cake is re-suspended using aprimer-probe cocktail to a 2× composition. The resulting mixture is usedto effect dissolution of the lyophilised master mix to a 2× solution.The complete solution will form a 2× mixture that may be further dilutedwith template to carry out a PCR.

1. A method for freeze-drying a reaction mixture comprising apolymerase, which comprises drying the reaction mixture with aglass-forming agent which is a polysaccharide having from 4 to 8saccharide units.
 2. The method of claim 1 wherein the saccharide isstachyose.
 3. (canceled)
 4. A composition comprising: (i) a polymerase;and (ii) a polysaccharide comprising from 4 to 8 saccharide units. 5.The composition of claim 4 which is in a freeze-dried form. 6.(canceled)
 7. The composition of claim 4 wherein the polymerase is athermostable polymerase.
 8. The composition of claim 4 wherein thepolymerase is a reverse-transciptase or is chemically modified orblocked so that it is inactivated until subject to an initial heatactivation step.
 9. (canceled)
 10. The composition of claim 4 whereinthe polysaccharide is stachyose.
 11. (canceled)
 12. The composition ofclaim 4 which is for carrying out a polymerase chain reaction and whichfurther comprises at least one of the following: (i) a buffer; (ii) ametal salt; (iii) nucleotides which may be fluorescently labelled; (iv)a blocking compound; (v) an anti-oxidant and/or anti-maillard reagent;(vi) a primer useful in a polymerase chain reaction which is optionallylabelled with a fluorescent label; (vii) a probe useful in detection ofa polymerase chain reaction which is optionally labelled with one ormore fluorescent labels; (viii) a fluorescent dye; (ix) an anti-Taqantibody that binds to the active site of the polymerase and inactivatesit at ambient temperature, but denatures and dissociates from the enzymeat elevated temperatures; (x) a pyrophosphate salt and thepyrophosphatase enzyme; (xi) a nucleic acid that is able to act as aninternal control for an amplification reaction; and (xii) a primer thatis able to amplify a nucleic acid of item (xi).
 13. The composition ofclaim 12 which is in the form of a freeze-dried cake and wherein atleast one of said further reagents is comprised within a separate layerwithin the cake.
 14. The composition of claim 4 which further comprisesa polymeric compound that is able to stabilize a freeze-dried cake.15-18. (canceled)
 19. A kit comprising the composition of claim 4 and asecond composition comprising a reagent for use in a reaction with saidpolymerase.
 20. The kit of claim 19 wherein the second composition is infreeze-dried form.
 21. The kit of claim 20 wherein said secondcomposition comprises stachyose and a reagent mixture for use in areaction with said polymerasee. 22-23. (canceled)
 24. The method ofclaim 1 for preparing a composition comprising a polymerase and apolysaccharide comprising from 4 to 8 saccharide units in freeze-driedform, which method comprises combining together the polymerase and thepolysaccharide and optionally one or more further components in solutionform, and freeze-drying said mixture.
 25. (canceled)
 26. The method ofclaim 24 wherein the polymerase is in the form of a solution from whichglycerol has been substantially removed.
 27. The method of claim 26wherein at least one of a detergent, an antioxidant and an anti-reducingagent is added to the polymerase after removal of the glycerol andbefore combination with the polysaccharide.
 28. The method of claim 24which comprises one or more further components and wherein the methodcomprises (i) freezing a solution of the polysaccharide and either oneor more of said further components or said polymerase to form a firstfrozen layer, (ii) adding a second solution of the polysaccharide as anda different one of said further components or said polymerase andfreezing the second solution into a distinct layer, (iii) optionallyrepeating step (ii) until all components of the composition are presentin one of the frozen layers, and (v) freeze-drying the resultantstructure.
 29. The method of claim 24 wherein the solution is dividedamongst multiple reaction vessels so that each vessel containssufficient reagents to carry out a single reaction using the polymerase,and freeze-dried within said vessels.
 30. (canceled)
 31. A method forcarrying out a chemical or biochemical reaction using a polymerase, saidmethod comprising forming a reaction mixture by addition of a liquid andany addition components necessary for carrying out said reaction to thecomposition of claim 4 in freeze-dried form, and subjecting the thusformed solution to suitable reaction conditions.
 32. The method of claim31 wherein the chemical or biochemical reaction is a polymerase chainreaction.